PROJECT SUMMARY The objective of the proposed research is to understand the functional role of Eph receptor clustering and signal transduction on neural stem cell function, harnessing a biomimetic strategy based on engineering synthetic multivalent ligands to modulate receptor complex formation. Cellular signal transduction often initiates with the multivalent interactions of ligands, either secreted or cell surface bound, to target cell receptors, leading to receptor clustering. Multivalent binding between ephrin and Eph receptors, which induces Eph clustering, has been found to play major roles in neurogenesis, immunology as well as the progression of cancer. However, current knowledge of the functional relationships between receptor clustering and signal activation, including how the latter depends on the number and spatial organization of receptors in a cluster, is limited. It was recently discovered that the interaction between EphB4 and eprhin-B2 regulates the neuronal differentiation of adult neural stem cells in the brain. Using this model system, I propose to engineer a series of novel multivalent EphB4 binding ligands to control the number, size, and nanoscale spatial organization of EphB4 receptors in a cluster. With this synthetic tool, I will investigate relationships between cluster properties, downstream signaling, and ultimately cell differentiation. Furthermore, the multivalent ligands that I will synthesize will serve as valuable reagents for elucidation of downstream signaling mechanisms. This work will have important implications for Eph signaling in many systems ? including neuroscience, immunology, and cancer biology ? as well as other receptor-ligand systems where oligomerization is critical for signaling.